Signaling in the retina and retinal pigment epithelium

National Eye Institute

Investigators

Linked publications & trials

Abstract

We are studying gene regulation and structural organization in RPE in the following ways: A) Analysis of RPE gene expression with special emphasis on differentiation/dedifferentiation pathways and protection against oxidative or inflammatory stress. Divergence from or convergence to the phenotype of native RPE is a common theme of RPE cell culture research and this has an important impact on the potential use of RPE cells in cell therapy for retinal degenerations. In particular, given the likely importance of noncoding RNAs as regulators of gene expression in the response of RPE cells to various signals, we are studying how lncRNA expression affects RPE differentiation and dedifferentiation, and how their expression may be altered with manipulation of RPE cells; B) Study of retina/RPE proteome. This is in 2 parts: i) the analysis of RPE organellar sub-proteomes; and ii) translational study of AMD plasma proteomics. The exact composition and stoichiometry of the visual cycle retinoid isomerization machinery (mostly located in the RPE smooth endoplasmic reticulum (ER)) is important to understanding this key RPE function. To answer this question, we plan to develop a complete catalog of the RPE smooth ER sub-proteome. Other complete RPE organellar sub-proteomes (lysosomes, melanosomes, mitochondria, etc.) will follow. These proteomes are most probably RPE-specific in particular ways, because of the specific functions of RPE, but are not completely known. This will be important to understanding the normal functions of RPE, as well as in RPE pathology such as AMD. With respect to AMD, we ask the question whether high resolution quantitative proteomics can detect changes in plasma proteins/peptides due to AMD. In the past year we have made progress in the following areas: 1) We continued a project studying the role of the lncRNA LINC00276 in RPE. RNASeq analysis provided a comprehensive view of differentially expressed lncRNAs in differentiated 4-month (4-mo) old ARPE-19 cells relative to 4-day (4-da) old cells. From these data, we observed a number of lncRNAs that were differentially regulated with fold change of 2.5 in differentiated ARPE-19 cells and which show a differential expression pattern between 4-da and 4-mo cultured cells. The expression of one of these in particular, LINC00276, was increased >200-fold in 4-mo cells compared to 4-da cells. Knockdown of LINC00276 negatively affected expression of various RPE-preferential transcripts, while its over-expression enhanced expression. By silencing LINC00276, we observed a decrease in the expression of genes associated with RPE differentiation such as MITF, TRPM1, TRPM3 and miR-204/211, while LINC00276 over-expression increased their expression. Silencing LINC00276 also decreased RPE-characteristic genes such as RPE65, TYR and MERTK, while altering the expression of genes involved in Wnt signaling pathway. We have determined that LINC00276 is preferentially expressed in native human RPE. We used RNA-pulldown and mass spectrometry analysis to identify proteins potentially interacting with this lncRNA and validated hits by western blotting and RNA immunoprecipitation assay. We identified the heterogeneous nuclear ribonucleoprotein L (HNRNPL) as being the major LINC00276-interacting protein in RPE. We used RNAscope multiplex nucleic acid in situ hybridization (ISH) assays to localize LINC00276 ncRNA, and HNRNPL and RPE65 mRNAs in ARPE-19 cells and human retina/RPE sections, and we observe a correlation in expression between these. The data obtained in this project are being written up for submission late in this reporting period or early in the next. 2) We initiated a project to determine how NIC-MEM medium, combined with N1, N21 or B27 supplementation, influences differentiation of the ARPE-19 proteome. ARPE-19 is a human RPE cell line with normal karyology, highly epithelial morphology, rapid rate of proliferation, and differentiation properties that distinguish them from primary RPE cultures. Suitable culture conditions promote differentiation, restore expression of genes and proteins, and functional and phenotypes resembling native RPE cells. For example, pyruvate, in combination with DMEM, induces dark pigmentation and transcription of differentiation markers such as CRALBP, MerTK, and RPE65, while adding nicotinamide (NIC) to MEM promotes ARPE-19 differentiation in alternate directions. We wished to determine how NIC-MEM medium, combined with N1, N21 and B27 supplementation, influences ARPE-19 proteome differentiation. We analyzed proteomes of ARPE-19 cells grown under different conditions. We found that NIC alone induces the upregulation of mitochondrial genes associated with the TCA cycle, respiratory electron transport, pyruvate metabolism, cristae formation, and mitochondria biogenesis. Pathways for oxidative phosphorylation, fatty acid metabolism, Myc targeting, mTORC1 signaling, and glycolysis are enhanced. Addition of N1 supplement to NIC-MEM medium contributes to the TCA cycle and mTORC1 signaling activation and N21 and B27 supplements induce a similar protein expression pattern in ARPE-19 cells. These supplements increase the expression of proteins related to the TCA cycle, oxidative phosphorylation, mTORC1 signaling, and amino acid metabolism. Also, N21 and B27 supplements induce the activation of Myc target genes and the inhibition of lysosomal proteins. Although, N1 supplement induced metabolic pathways related to ARPE-19 differentiation, the biggest effects were obtained when the cells grew in NIC-MEM medium supplemented with N21 and B27. We concluded that NIC, N1, N21, and B27 affect ARPE-19 cellular processes in distinct ways. These analyses confirm that ARPE-19 is a highly plastic cell line that responds directly to small changes in its growing conditions. As in vivo pathologic changes, such as from proliferative vitreoretinopathy, AMD, etc., affect RPE cellular milieu, our experiments provide a model for analysis of different effectors on RPE, including disease. We are applying this proteomics analysis to such conditions. These data are being readied for publication and a manuscript will be submitted late in this reporting period or early in the next. 3) We continued a project (lead investigator: Dr. Gleysin Cabrera) to determine if AMD can be detected using quantitative proteomics (QP). This project is being done in collaboration with Dr. Emily Chew, DECA, and involves analysis of plasma samples from subjects with different stages of AMD collected under the AREDS protocol. Our hypothesis is that eyes at risk of GA/AMD initiation or progression may secrete proteins/proteoforms into the bloodstream that can be detected as biomarkers. These may be present at very low levels but modern QP could bridge this gap. We will use Seer Proteograph sera/plasma QP to identify biomarkers that could then be used for early and accurate diagnosis of GA/AMD. Viable protein markers from these analyses will then be analyzed by targeted parallel reaction monitoring (PRM)-mass spectrometry and tested on a validation cohort of subjects of unknown diagnosis. Serum samples from age-related macular degeneration (AMD) patients (20 total) in different AREDS severity grades (SG1-SG4) were used for these proteomics studies. Proteomics data was acquired by LC-MS and MS data was processed using DIA-NN (v.1.8.1) using standard settings and workflow and more than 2400 proteins per serum sample were identified. GO (Gene Ontology) and pathways enrichment analysis were performed using Seer Proteograph Analysis Suite (PAS), DIA-Analyst, Enrichr, gProfiler, SRplot, String, and Metascape. This proteomic study identified the differential expression of 17 proteins encoded by loci strongly associated with AMD (out of 34 loci) in genome-wide association studies (GWAS) suggesting serum could be a good sample for biomarker discovery in AMD. Pathways identified in the different stages include complement cascade, scavenger receptor, TCA cycle, activation of the Ras-Raf-MEK-ERK pathway, platelet activation. In the later stages, there was downregulation in several GO biological processes, component compartments and molecular functions related to chromatin organization, including histones and other factors, and resulting in epigenetic changes. Certain factors are downregulated at all stages. These analyses are currently being repeated and analyzed together with other analyses being performed by DECA (Dr. Emily Chew and colleagues). A manuscript is being prepared and will be submitted in the next reporting period. 4) We continue to collaborate with sections in the LRCMB and with other NEI laboratories and sections (e.g., Molecular Structure and Functional Genomics, Neurobiology-Neurodegeneration Repair Laboratory, Laboratory of Immunology, etc.), as well as with extramural labs in the analysis (HPLC and mass spectrometry) of proteins, retinoids, lipids, and other compounds. Several separate collaborative studies are ongoing.